Iron is the most common transition metal found in proteins and participates in many fundamental physiological functions in all living organisms, in particular in processes related to energy conservation. Among the iron-containing structures which are common to eucaryotes as well as procaryotes, iron-sulfur clusters are extremely important, not only because of their ubiquity, but also because of their unique redox properties. The mechanisms of incorporation and stabilization of iron into these structures is still far from being well understood. Anerobic bacteria, which are important to man in disease and in health, constitute an excellent source of iron-sulfur cluster containing proteins and, as such, are ideal tools for their study. The long term objective of this research project is to understand the conversion mechanisms between three and four iron-containing clusters found in certain ferredoxins and their significance for the regulation of electron flow in multiple electron transfer chains. The use of ferredoxins and artificially reconceived polypeptides as templates for the synthesis of new mixed-metal clusters will also be explored as a means to develop new molecules and examine their potential catalytic properties. Specific aims of this research are: 1) to prepare new mixed-metal clusters incorporated into Desulfovibrio gigas ferredoxin II (a Co-Fe and a Zn-Fe cluster have already been obtained from this protein; 2) to search for other ferredoxins that could perform better than the D. gigas protein in the synthesis of these new metal clusters; 3) to prepare specifically isotope-labelled, either 57 Fe or 56 Fe iron-sulfur clusters and investigate their properties using different spectrometric techniques; 4) to clone and express D. gigas ferredoxin gene in E. coli in order to over-produce the protein for physical studies and also to be able to perform site-directed mutagenesis to understand how 3Fe and 4Fe sulfur clusters are formed and stabilized; 5) to perform physiological experiments with the transformed mixed-metal clusters and the mutated proteins; 6) to extend these techniques to complex metal-containing enzymes; 7) to monitor the above experiments using modern computer graphics modeling.